Methods of determining potency of chemically-synthesized oligonucleotides

ABSTRACT

Provided herein are methods for determining potency of RNAi agents. Such methods include, but are not limited to, cell-based and cell-free assays that measure binding of an RNAi agent with Ago2 or that measure Ago2 activity in the presence of such RNAi agents. Also provided are assays that determine potency of RNAi agents by assessing their ability to compete with other RNAi agents, including control RNAi agents, for binding and/or activation of Ago2.

FIELD OF THE INVENTION

The present invention is drawn to methods of determining the potency ofone or more chemically synthesized RNAi agents.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is a mechanism by which double-stranded RNAtriggers the silencing of target gene expression by inducingsequence-specific target mRNA degradation. In certain instances, shortinterfering RNAs (siRNAs), dsRNA duplexes of 21-23 nucleotides, aremediators in the RNAi pathway that lead to degradation of specific mRNAsthrough the RNA induced silencing complex (RISC). Once introduced intocells, siRNA molecules bind to Argonaute2, a component of RISC, thatcatalyzes the cleavage of the target mRNA.

SUMMARY OF THE INVENTION

Here we show that siRNA competition, which varies significantly betweencell lines, is correlated to differences in the expression levels ofAgo2. While cellular Ago2 levels have dramatic effects on siRNAcompetition and potency, levels of other RISC-associated proteins donot. We further demonstrate the role of Ago2 in siRNA competition andpotency by overexpression and siRNA mediated reduction of Ago2 withincells. In addition, we show that when Ago2 is limiting, siRNAs withhigher affinity for Ago2 are favored for RISC loading due to morefavorable binding affinity for purified Ago2. Finally, siRNA competitionwas used to analyze the kinetics of RISC loading and unloading. Aftertransfection, RISC loading occurs in approximately 2 hours, then siRNAsremain associated with RISC for 8-12 hours, but new protein synthesis isnot required to generate active RISC.

Therefore, the present invention is drawn to methods of determining therelative potency of one or more chemically synthesized RNAi agents bymeasuring the binding efficiencies of one or more chemically synthesizedRNA; agents to human eukaryotic translation initiation factor 2C, 2(eIF2C2) (Argonaut 2 or Ago2), Accession No. NP_(—)036286, which isherein incorporated by reference. Potency as contemplated herein is theRNAi agent's ability to activate RISC and reduce the amount of targetRNA to which the RNAi agent is designed. In an embodiment of theinvention a labeled target RNA segment can be introduced into the invitro system to measure the amount of the labeled target RNA segment iscleaved by the activated RNAi agent-activated RISC. In addition, thepresent invention contemplates determining the relative potency of oneor more compounds in an in vitro assay comprising either recombinanthuman Ago2 or immunoprecipitated Ago2. The types of binding assays thatcould be useful in the context of the present invention would includetechniques well known by those of ordinary skill in the art indetermining potency as indicated by binding affinity. These techniquesinclude, but are not limited to, homologous saturation, competition,activity (competition), gel shift, filter binding, or size-exclusionchromatography. An additional embodiment of the invention would also bethe use of in silico screening of test RNAi agents to determine bindingefficiency to Ago2 and therefore the test RNAi agent's potency.

The methods disclosed herein are useful in the developing a structureactivity relationship for chemically-synthesized RNAi agents. In anembodiment of the invention the RNAi agents are antisenseoligonucleotides which have one or more sugar-modified nucleotide,modified internucleoside linkage, or one modified nucleobase. In acertain embodiment, the sugar modified nucleotides are 2′-modifiednucleotides or bicyclic nucleotides. In an additional embodiment, themodified internucleoside linkages are phosphorothioates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Magnitude of PTEN and Eg5 siRNA competition varies between celllines. Cells were treated at doses 0.02 to 20 nM Eg5 siRNA in thepresence of PTEN competitor siRNA at a dose of 2, 6, or 20 nM for 4hours. The following day qRT/PCR was performed to assess the reductionof Eg5 mRNA. IC₅₀ curves are shown for Eg5 siRNA mediated mRNA reductionin the presence of 0 (▪, 2(▾), 6 (), or 20 (Δ) nM PTEN siRNA. A) HeLacells. B) T47D cells. C) U87-MG cells. Calculated IC₅₀'s with standarderror for each Eg5 mRNA inhibition are shown at the bottom of thefigure.

FIG. 2. RISC reduction and overexpression A) RISC mRNA reduction byRNAseH dependent antisense oligonucleotides and siRNAs. HeLa cells weretreated with siRNAs or ASOs targeted to Dicer, Ago2, TRBP or exportin-5at 50 nM. After 24 hours, cells were harvested and expression of thetargeted mRNA determined by qRT/PCR. The results shown are the percentcontrol relative to untreated cells. B) RISC overexpression. HeLa cellswere transfected with mammalian expression plasmids for human Dicer,Ago2, TRBP, or exportin-5. After 24 hours cells were harvested andlysates prepared for analysis of protein expression by Western blot.Duplicate lanes were loaded with 15 ug each of total protein. TubulinmAb was used as a loading control.

FIG. 3: Ago2 reduction effects both potency and magnitude of siRNAcompetition. HeLa cells were treated with siRNAs targeting Ago2, Diceror XPO5. After 24 hours cells were seed in 96 well plates then treated 4hours later cells at Eg5 siRNA doses from 2 μM to 60 nM in the presenceor absence of 10 nM PTEN competitor siRNA for 4 hours. The following dayIC₅₀s for Eg5 mRNA reduction were generated by qRT/PCR. A) Levels ofAgo2 and Dicer reduction in siRNA treated cells were assessed by Westernlot 24 hours after initiation of treatment. B) Eg5 IC₅₀ curves for HeLacells in the presence (solid line) or absence (dotted line) of PTENcompetitor siRNA. C) Levels of Ago2 and XPO5 reduction in siRNA treatedcells were assessed by Western blot 24 hours after initiation oftreatment. D) Eg5 IC₅₀ curves for HeLas in the presence (solid line) orabsence (dotted line) of PTEN competitor siRNA. Eg5 IC₅₀s with standarderror are shown in the absence (▪) or presence (▴) of PTEN siRNAcompetitor. The fold change in IC₅₀ for each cell line is shown at thebottom of the figure.

FIG. 4. siRNA potency and competition are related to relative affinitiesfor Ago2. A) Competitive binding of Eg5 and PTEN siRNA guide strands topurified Ago2. B) IC₅₀'s in U87-Mg cells for PTEN siRNA competed withEg5 siRNA at 0, 2, 6 and 20 nM as in FIG. 2.

FIG. 5. Kinetics of RISC loading and unloading. A) RISC loading. HeLacells were treated with Eg5 siRNA at 300 pM. 10 nM PTEN siRNA competitorwas added between 0 and 240 minutes after the initiation of the Eg5siRNA transfection. Transfections were carried out for a total of 5hours. Cells were harvested the following day and total RNA purified.The percent inhibition of Eg5 mRNA is shown at the various timepointsfor the addition of PTEN competitor. E=Eg5 siRNA only, no competitor. B)RISC unloading. HeLa cells were treated for 3 hours with PTEN siRNA.Cells were washed then treated with 300 pM Eg5 siRNA at 0 to 18 hoursfollowing the removal of the PTEN siRNA. Transfections were carried outfor 3 hours. The following day cells were harvested and total RNApurified. The percent inhibition of Eg5 mRNA is shown at the varioustimepoints following removal of PTEN competitor siRNA. The experimentwas performed in the presence (solid bars) or absence (striped bars) of25 μg/ml cycloheximide (CHX).

FIG. 6. Kinetics of mRNA reduction by Eg5 siRNA. HeLa cells weretransfected with 10 nM Eg5 siRNA as detailed in Materials and Methods.siRNA treated and control cells were harvested and Eg5 mRNA reductiondetermined at the indicated times by qRT/PCR.

Tables

TABLE I Control Ago2 10 nM PTEN siRNA − + − + IC₅₀ (pM) 291 ± 79 1640 ±499 65 ± 18 138 ± 40 Fold Change IC₅₀ 5.6 2.1 Overexpression of Ago2 inU87-MG cells affects siRNA potency and competition. U87-MG cells weretransfected with a pCMV-Ago2. After 24 hours Ago2 overexpression wasconfirmed by Western blot and cells were seeded in 96 well plates thentreated with Eg5 siRNA at doses from 2 pM to 60 nM (N = 4/dose) in thepresence or absence of 10 nM PTEN competitor siRNA for 4 hours. Thefollowing day IC₅₀s for Eg5 mRNA reduction were generated by qRT/PCR.Calculated IC₅₀'s with standard error for control and Ago2overexpressing cells, along with the fold change in IC₅₀ in the presenceof competitor are shown.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed. Herein, the use ofthe singular includes the plural unless specifically stated otherwise.As used herein, the use of “or” means “and/or” unless stated otherwise.Furthermore, the use of the term “including” as well as other forms,such as “includes” and “included”, is not limiting. Also, terms such as“element” or “component” encompass both elements and componentscomprising one unit and elements and components that comprise more thanone subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

A. Definitions

Unless specific definitions are provided, the nomenclature utilized inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, andchemical analysis. Certain such techniques and procedures may be foundfor example in “Carbohydrate Modifications in Antisense Research” Editedby Sangvi and Cook, American Chemical Society, Washington D.C., 1994;“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,18th edition, 1990; and “Antisense Drug Technology, Principles,Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press,Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratoryManual,” 2^(nd) Edition, Cold Spring Harbor Laboratory Press, 1989;which are hereby incorporated by reference for any purpose. Wherepermitted, all patents, applications, published applications and otherpublications and other data referred to throughout in the disclosureherein are incorporated by reference in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, the term “Ago2” refers to a protein having Accession No.NP_(—)36286 and variants thereof.

As used herein, the term “binding affinity” refers to the associationbetween two molecules. Binding is typically expressed as K_(d). Incertain embodiments, binding affinity refers to the association of anRNAi agent with Ago2.

As used herein, the term “Ago2 binding affinity” refers to the bindingaffinity of a molecule with Ago2.

As used herein, the term “RNAi” refers to modulation of a target nucleicacid or target protein through RISC.

As used herein, the term “RISC” refers to the RNA induced silencingcomplex.

As used herein, the term “RNAi agent” refers to any molecule thatactivates the RISC pathway. RNAi agents include, but are not limited tosiRNA and asRNA.

As used herein, the term “RNAi activity” refers to the ability of anRNAi agent to effect cleavage of a target nucleic acid.

As used herein, the term “Ago2 activity” refers to the ability of anRNAi agent to activate Ago2 to effect cleavage of a target nucleic acid.In certain embodiments, such Ago2 activity may be in a cell or may be ina cell free assay.

As used herein, the term “RNAi potency” refers to the ability of an RNAiagent to effect cleavage of a target nucleic acid or to otherwisemodulate the amount of target protein in a cell or in an animal. Suchcleavage or protein modulation may be measured directly or indirectly.

As used herein, the term “siRNA” refers to an RNAi agent that is adouble stranded oligonucleotide.

As used herein, the term “asRNA” refers to an RNAi agent that is asingle stranded oligonucleotide.

As used herein, the term “oligonucleotide” refers to a compoundcomprising a plurality of linked nucleotides or nucleosides. In certainembodiment, one or more nucleotides of an oligonucleotide is modified.In certain embodiments, an oligonucleotide comprises ribonucleic acid(RNA) or deoxyribonucleic acid (DNA). In certain embodiments,oligonucleotides are composed of natural and/or modified nucleobases,sugars and covalent internucleoside linkages, and may further includenon-nucleic acid conjugates.

As used herein, the term “nucleoside” means a glycosylamine comprising anucleobase and a sugar. Nucleosides includes, but are not limited to,natural nucleosides, a basic nucleosides, modified nucleosides, andnucleosides having mimetic bases and/or sugar groups.

As used herein, the term “natural nucleoside” or “unmodified nucleoside”means a nucleoside comprising a natural nucleobase and a natural sugar.Natural nucleosides include RNA and DNA nucleosides.

As used herein, the term “natural sugar” refers to a sugar of anucleoside that is unmodified from its naturally occurring form in RNA(2′-OH) or DNA (2′-H).

As used herein, the term “nucleobase” refers to the base portion of anucleoside or nucleotide. A nucleobase may comprise any atom or group ofatoms capable of hydrogen bonding to a base of another nucleic acid.

As used herein, the term “natural nucleobase” refers to a nucleobasethat is unmodified from its naturally occurring form in RNA or DNA.

As used herein, the term “heterocyclic base moiety” refers to anucleobase comprising a heterocycle.

As used herein “oligonucleotide” refers to an oligonucleotide in whichthe internucleoside linkages do not contain a phosphorus atom.

As used herein “internucleoside linkage” refers to a covalent linkagebetween adjacent nucleosides.

As used herein “natural internucleotide linkage” refers to a 3′ to 5′phosphodiester linkage. As used herein, the term “modifiedinternucleoside linkage” refers to any linkage between nucleosides ornucleotides other than a naturally occurring internucleoside linkage.

As used herein, the term “antisense compound” refers to an oligomericcompound that is at least partially complementary to a target nucleicacid molecule to which it hybridizes. In certain embodiments, anantisense compound modulates (increases or decreases) expression of atarget nucleic acid.

As used herein, the term “antisense oligonucleotide” refers to anantisense compound that is an oligonucleotide.

As used herein, the term “antisense activity” refers to any detectableand/or measurable activity attributable to the hybridization of anantisense compound to its target nucleic acid. Such detection and ormeasuring may be direct or indirect. For example, in certainembodiments, antisense activity is assessed by detecting and ormeasuring the amount of target protein. In certain embodiments,antisense activity is assessed by detecting and/or measuring the amountof target nucleic acids and/or cleaved target nucleic acids and/oralternatively spliced target nucleic acids. In certain embodiments,antisense activity is mediated by RNase H, by RISC or by interferingwith normal splicing of a pre-mRNA.

As used herein the term “detecting RNAi activity” or “measuring RNAiactivity” means that a test for detecting or measuring RNAi activity isperformed on a sample. Such detection and/or measuring may includevalues of zero. Thus, if a test for detection of RNAi activity resultsin a finding of no RNAi activity (RNAi activity of zero), the step of“detecting RNAi activity” has nevertheless been performed.

As used herein the term “control sample” refers to a sample that has notbeen contacted with a test compound. In certain embodiments, a controlsample is obtained prior to administration of a compound to an animal.In certain embodiments, a control sample is obtained from an animal towhich compound is not administered. In certain embodiments, a referencestandard is used as a surrogate for a control sample.

As used herein the term “chimeric oligonucleotide” refers to anoligonucleotide, having at least one sugar, nucleobase and/orinternucleoside linkage that is differentially modified as compared tothe other sugars, nucleobases and internucleoside linkages within thesame oligonucleotide. The remainder of the sugars, nucleobases andinternucleoside linkages can be independently modified or unmodified. Ingeneral a chimeric oligonucleotide will have modified nucleosides thatcan be in isolated positions or grouped together in regions that willdefine a particular motif. Any combination of modifications and ormimetic groups can comprise a chimeric oligomeric compound as describedherein.

As used herein, the term “motif” refers to a pattern of unmodified andmodified nucleotides or linkages in an oligonucleotide.

As used herein, the term “mixed-backbone oligonucleotide” refers to anoligonucleotide wherein at least one internucleoside linkage of theoligonucleotide is different from at least one other internucleotidelinkage of the oligonucleotide.

As used herein, the term “target protein” refers to a protein, themodulation of which is desired.

As used herein, the term “target gene” refers to a gene encoding atarget.

As used herein, the term “target nucleic acid” refers to any nucleicacid molecule, the amount or function of which is capable of beingmodulated. Target nucleic acids include, but are not limited to, RNA(including, but not limited to pre-mRNA and mRNA or portions thereof),cDNA derived from such RNA, as well as non-translated RNA, such asmiRNA. For example, in certain embodiments, a target nucleic acid can bea cellular gene (or mRNA transcribed from such gene) whose expression isassociated with a particular disorder or disease state, or a nucleicacid molecule from an infectious agent.

As used herein, the term “targeting” or “targeted to” refers to theassociation of an antisense compound to a particular target nucleic acidmolecule or a particular region of nucleotides within a target nucleicacid molecule.

As used herein, “designing” or “designed to” refer to the process ofdesigning an oligomeric compound that specifically hybridizes with aselected target nucleic acid molecule.

As used herein, the term “nucleobase complementarity” refers to anucleobase that is capable of base pairing with another nucleobase. Forexample, in DNA, adenine (A) is complementary to thymine (T). Forexample, in RNA, adenine (A) is complementary to uracil (U). In certainembodiments, complementary nucleobase refers to a nucleobase of anantisense compound that is capable of base pairing with a nucleobase ofits target nucleic acid. For example, if a nucleobase at a certainposition of an antisense compound is capable of hydrogen bonding with anucleobase at a certain position of a target nucleic acid, then theposition of hydrogen bonding between the oligonucleotide and the targetnucleic acid is considered to be complementary at that nucleobase pair.

As used herein, the term “non-complementary nucleobase” refers to a pairof nucleobases that do not form hydrogen bonds with one another orotherwise support hybridization.

As used herein, the term “complementary” refers to the capacity of anoligomeric compound to hybridize to another oligomeric compound ornucleic acid through nucleobase complementarity. In certain embodiments,an antisense compound and its target are complementary to each otherwhen a sufficient number of corresponding positions in each molecule areoccupied by nucleobases that can bond with each other to allow stableassociation between the antisense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are antisense compounds thatmay comprise up to about 20% nucleotides that are mismatched (i.e., arenot nucleobase complementary to the corresponding nucleotides of thetarget). Preferably the antisense compounds contain no more than about15%, more preferably not more than about 10%, most preferably not morethan 5% or no mismatches. The remaining nucleotides are nucleobasecomplementary or otherwise do not disrupt hybridization (e.g., universalbases). One of ordinary skill in the art would recognize the compoundsprovided herein are at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%nucleobase complementary to a target nucleic acid.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases). For example,the natural base adenine is nucleobase complementary to the naturalnucleobases thymidine and uracil which pair through the formation ofhydrogen bonds. The natural base guanine is nucleobase complementary tothe natural bases cytosine and 5-methyl cytosine. Hybridization canoccur under varying circumstances.

As used herein, the term “specifically hybridizes” refers to the abilityof an oligomeric compound to hybridize to one nucleic acid site withgreater affinity than it hybridizes to another nucleic acid site. Incertain embodiments, an antisense oligonucleotide specificallyhybridizes to more than one target site. In certain embodiments, anoligomeric compound specifically hybridizes with its target understringent hybridization conditions.

As used herein, the term “stringent hybridization conditions” or“stringent conditions” refers to conditions under which an antisensecompound will hybridize to its target sequence, but to a minimal numberof other sequences. Stringent conditions are sequence-dependent and willbe different in different circumstances, and “stringent conditions”under which antisense compounds hybridize to a target sequence aredetermined by the nature and composition of the antisense compounds andthe assays in which they are being investigated.

As used herein, the term “modulation” refers to a perturbation offunction or activity when compared to the level of the function oractivity prior to modulation. For example, modulation includes thechange, either an increase (stimulation or induction) or a decrease(inhibition or reduction) in gene expression. As further example,modulation of expression can include perturbing splice site selection ofpre-mRNA processing.

As used herein, the term “expression” refers to all the functions andsteps by which a gene's coded information is converted into structurespresent and operating in a cell. Such structures include, but are notlimited to the products of transcription and translation.

As used herein, “high-affinity modified monomer” refers to a monomerhaving at least one modified nucleobase, internucleoside linkage orsugar moiety, when compared to naturally occurring monomers, such thatthe modification increases the affinity of an antisense compoundcomprising the high-affinity modified monomer to its target nucleicacid. High-affinity modifications include, but are not limited to,monomers (e.g., nucleosides and nucleotides) comprising 2′-modifiedsugars.

As used herein, the term “2′-modified” or “2′-substituted” means a sugarof a nucleoside comprising a substituent at the 2′ position other than Hor OH.

As used herein, the term “MOE” refers to a 2′-O-methoxyethylsubstituent.

As used herein, the term “high-affinity modified nucleotide” refers to anucleotide having at least one modified nucleobase, internucleosidelinkage or sugar moiety, such that the modification increases theaffinity of an antisense compound comprising the modified nucleotide toa target nucleic acid. High-affinity modifications include, but are notlimited to, BNAs, LNAs and 2′-MOE.

As used herein the term “mimetic” refers to groups that are substitutedfor a sugar, a nucleobase, and/or internucleoside linkage. Generally, amimetic is used in place of the sugar or sugar-internucleoside linkagecombination, and the nucleobase is maintained for hybridization to aselected target. Representative examples of a sugar mimetic include, butare not limited to, cyclohexenyl or morpholino. Representative examplesof a mimetic for a sugar-internucleoside linkage combination include,but are not limited to, peptide nucleic acids (PNA) and morpholinogroups linked by uncharged achiral linkages. In some instances a mimeticis used in place of the nucleobase. Representative nucleobase mimeticsare well known in the art and include, but are not limited to, tricyclicphenoxazine analogs and universal bases (Berger et al., Nuc Acid Res.2000, 28:2911-14, incorporated herein by reference). Methods ofsynthesis of sugar, nucleoside and nucleobase mimetics are well known tothose skilled in the art.

As used herein, the term “bicyclic nucleic acid” or “BNA” refers to anucleoside wherein the furanose portion of the nucleoside includes abridge connecting two atoms on the furanose ring, thereby forming abicyclic ring system. BNAs include, but are not limited to, α-L-LNA,β-D-LNA, ENA, Oxyamino BNA (2′-O—N(CH₃)—CH₂-4′) and Aminooxy BNA(2′-N(CH₃)—O—CH₂-4′).

As used herein, a “locked nucleic acid” or “LNA” refers to a nucleotidemodified such that the 2′-hydroxyl group of the ribosyl sugar ring islinked to the 4′ carbon atom of the sugar ring, thereby forming a2′-C,4′-C-oxymethylene linkage. LNAs include, but are not limited to,α-L-LNA, and β-D-LNA.

As used herein, the term “cap structure” or “terminal cap moiety” refersto chemical modifications, which have been incorporated at eitherterminus of an antisense compound.

As used herein, the term “animal” refers to a human or non-human animal,including, but not limited to, mice, rats, rabbits, dogs, cats, pigs,and non-human primates, including, but not limited to, monkeys andchimpanzees.

Overview

In certain embodiments, the present invention provides methods ofpredicting potency of an RNAi agent. In certain embodiments, suchmethods comprise determining the binding affinity of the RNAi agent withAgo2. In certain embodiments, such methods comprise determining the Ago2activity of an RNAi agent. In certain embodiments, such determinationsare made relative to a control and/or a reference standard. In certainembodiments, relative potency of two RNAi agents is predicted orassessed. In certain such embodiments, a competition assay is performed,wherein two RNAi agents are in competition with Ago2 and Ago2 bindingand/or Ago2 activity is measured.

In certain embodiments the amount of Ago2 or the amount of Ago2 activitypresent in a cell is the rate limiting factor in RNAi activity. Incertain cell types, Ago2 is the least abundant protein component of theRISC pathway. In certain embodiments, suppression of other members ofthe RISC pathway has little or no effect on RNAi activity. Accordingly,in certain such embodiments, RNAi activity correlates with Ago2 activityand/or with Ago2 binding. Thus, in certain embodiments, measuring Ago2binding and/or Ago2 activity predicts RNAi activity and RNAi potency ina cell or tissue for a particular RNAi agent.

In certain embodiments, Ago2 binding of an RNAi agent is assessed. Incertain such embodiments, Ago2 binding of an RNAi agent is assessed in acell. In certain such embodiments, an RNAi agent is contacted with acell to allow it to bind to Ago2 inside the cell; Ago2 is thenprecipitated from the cell and binding of the RNAi agent with the Ago2is assessed. In certain embodiments, Ago2 binding of an RNAi agent isassessed in a cell-free assay. In certain such embodiments, Ago2 iscontacted with an RNAi agent. In certain such embodiments, the Ago2 isobtained from immunoprecipitation from a cell. In certain suchembodiments, Ago2 is overexpressed in the cell prior toimmunoprecipitation. In certain embodiments the Ago2 is recombinant. Incertain such embodiments, the recombinant Ago2 is isolated and purified.In certain embodiments, Ago2 binding of an RNAi agent correlates withAgo2 activity, with RNAi activity and/or RNAi potency. Accordingly, suchAgo2 binding determination may be used to predict Ago2 activity, RNAiactivity, and/or RNAi potency.

In certain embodiments, Ago2 activity of an RNAi agent is assessed. Incertain such embodiments, Ago2 activity of an RNAi agent is assessed ina cell. In certain such embodiments, an RNAi agent is contacted with acell to allow it to bind to Ago2 inside the cell; Ago2 is thenprecipitated from the cell and Ago2 activity is assessed. In certainsuch embodiments, Ago2 activity is assessed by contacting theprecipitated Ago2/RNAi agent complex with a substrate RNA and detectingcleavage of the RNA substrate. In certain embodiments, Ago2 activity ofan RNAi agent is assessed in a cell free assay. In certain suchembodiments, Ago2 is contacted with an RNAi agent and with a substrateRNA and cleavage of the substrate RNA is detected. In certain suchembodiments, the Ago2 is obtained from immunoprecipitation from a cell.In certain such embodiments, Ago2 is overexpressed in the cell prior toimmunoprecipitation. In certain embodiments the Ago2 is recombinant. Incertain such embodiments, the recombinant Ago2 is isolated and purified.In certain embodiments, Ago2 activity of an RNAi agent correlates withAgo2 binding, with RNAi activity and/or RNAi potency. Accordingly, suchAgo2 activity determination may be used to predict Ago2 binding, RNAiactivity, and/or RNAi potency.

In certain embodiments, the present invention provides competitionassays. In certain such embodiments, assays to detect Ago2 binding orAgo2 activity described above are performed with two RNAi agents. Incertain embodiments, such assays include test samples of varyingconcentrations of one or both of the RNAi agents. In certainembodiments, one of the RNAi agents in a competition assay is an RNAiagent of known Ago2 binding, Ago2 activity, and/or Ago2 potency (knowncompetitor). In certain such embodiments, Ago2 binding or Ago2 activityof the known competitor is assessed. In certain embodiments, the abilityof a test RNAi agent to inhibit Ago2 binding or Ago2 activity of theknown competitor indicates that it has desirable binding, activity,and/or potency. In certain embodiments, the invention provides an assaysystem whereby two or more test RNAi agents are separately tested fortheir ability to compete against the same known competitor. In suchembodiments, binding or activity of the two or more test RNAi agents maybe compared by comparing their ability to inhibit binding or activity ofthe known competitor.

In certain such embodiments, (1) cells or Ago2 (from immunoprecipitationor recombinant) is placed in a multiwell plate; (2) a known competitorRNAi agent is added to each well at the same concentration per well; (3)a test RNAi agent is added to each of several wells, typically atseveral different concentrations; and (4) Ago2 activity or binding ofeither the known competitor RNAi agent or the test RNAi agent or both isdetected and/or measured. In certain embodiments, such assays are usefulfor assessing the relative RNAi activity or potency of the test RNAiagent compared to the known competitor RNAi agent or compared to anothertest RNAi agent that is tested using the same known competitor RNAiagent. In certain embodiments, the concentration of the known competitorRNAi agent is varied and the concentration of the test RNAi agent is thesame for each well. In certain embodiments, two or more competitoroligomeric compounds are separately tested against the same knowncompetitor RNAi agent to assess the relative uptake of the two or moretest RNAi agents. In certain embodiments, the known competitor RNAiagent is replaced with a second test RNAi agent. In such embodiments,one may measure or detect binding of one or both of the test RNAiagents. One of ordinary skill in the art will readily appreciate thatthese components can be manipulated in a variety of ways. Certaincompetition assays have been described previously. See e.g., Koller etal., Nucleic Acid Research, 34:16, 4467-4476 (2006), which is herebyincorporated by reference in its entirety.

Certain RNAi Agents

In certain embodiments, the present invention provides methods ofpredicting or assessing RNAi activity and/or RNAi potency of an RNAiagent. Such methods may be performed using any molecule suspected ofhaving RNAi activity. In certain embodiments, an RNAi agent may be asynthetic small molecule or a peptide. In certain embodiments, RNAiagents are oligonucleotides. Such oligonucleotides may be singlestranded or they may be double stranded. RNAi agents may compriseoligonucleotides that are modified. In the case of double-strandedoligonucleotide RNAi agents, one or both strands may be modified. Ifonly one strand of a double stranded RNAi agent is modified, themodified strand may be a sense strand or it may be an antisense strand.

Certain RNAi agents have greater RNAi activity and RNAi potency thanothers. Certain siRNAs and as RNAs likewise have greater RNAi activityand RNAi potency than others. Such differences may be attributable todifferences in length, chemical modification, sequence, target, or acombination of these and other factors.

In certain instances, it appears that the difference in potency isattributable to sequence. In certain competition assays, certain siRNAsdemonstrate greater ability to compete. For example, when a PTEN siRNAis co-administered with an Eg5 siRNA, the PTEN siRNA consistently led tothe degradation of PTEN mRNA and reduced the degradation of Eg5 RNAdirected by the Eg5 siRNA. Both siRNAs were blunt-ended 19mer duplexesof unmodified RNA, transfected simultaneously into the same cells, sothe competitive advantage enjoyed by the PTEN siRNA must be due to itssequence relative to the Eg5 sequence. Thus, in certain embodiments, thepresent invention provides a competition assay to identify and/orpredict potent RNAi agents based on their ability to compete withanother RNAi agent. In certain embodiments, the two RNAi agents testedin a competition assay have the same length and motif. In certainembodiments, RNAi agents tested in a competition assay have one or moredifferences in length and motif.

In certain embodiments, the present invention provides methods forpredicting or assessing RNAi activity of single or double strandedoligonucleotides comprising different motifs. In certain embodiments,the present invention provides methods for predicting or assessing RNAiactivity of single or double stranded oligonucleotides comprisingdifferent modifications. Such motifs and modifications include, but arenot limited to, oligonucleotides comprising modified bases, modifiedinternucleoside linkages and modified sugars.

In certain embodiments, an RNAi agent comprises a single or doublestranded oligonucleotide comprising one or more modified nucleosidecomprising a modified sugar. In such embodiments, the furanosyl sugarring of the nucleoside can be modified in a number of ways including,but not limited to, addition of a substituent group, bridging of twonon-geminal ring atoms to form a bicyclic nucleic acid (BNA) andsubstitution of an atom or group such as —S—, —N(R)— or —C(R₁)(R₂) forthe ring oxygen at the 4′-position.

BNA's have been prepared and disclosed in the patent literature as wellas in scientific literature (Singh et al., Chem. Commun., 1998, 4,455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt etal., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al.,Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; WO 94/14226; WO2005/021570; Singh et al., J. Org. Chem., 1998, 63, 10035-10039;Examples of issued US patents and published applications that discloseBNAs include, for example, U.S. Pat. Nos. 7,053,207; 6,268,490;6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-GrantPublication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841;2004-0143114; and 20030082807.

In certain embodiments, RNAi agents may comprise one or more “LockedNucleic Acids” (LNAs) nucleosides, in which the 2′-hydroxyl group of theribosyl sugar ring is linked to the 4′ carbon atom of the sugar ringthereby forming a 2′-C,4′-C-oxymethylene linkage to form the bicyclicsugar moiety (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs,2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8 1-7; and Orum etal., Curr. Opinion Mol. Ther., 2001, 3, 239-243; see also U.S. Pat. Nos.6,268,490 and 6,670,461). The linkage can be a methylene (—CH₂—) groupbridging the 2′ oxygen atom and the 4′ carbon atom, for which the termLNA is used for the bicyclic moiety; in the case of an ethylene group inthis position, the term ENA™ is used (Singh et al., Chem. Commun., 1998,4, 455-456; ENA™: Morita et al., Bioorganic Medicinal Chemistry, 2003,11, 2211-2226). LNA and other bicyclic sugar analogs display very highduplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10°C.), stability towards 3′-exonucleolytic degradation and good solubilityproperties. Potent and nontoxic antisense oligonucleotides containingLNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 5633-5638).

An isomer of LNA that may be used in certain embodiments, is alpha-L-LNAwhich has been shown to have superior stability against a3′-exonuclease. The alpha-L-LNA's were incorporated into antisensegapmers and chimeras that showed potent antisense activity (Frieden etal., Nucleic Acids Research, 2003, 21, 6365-6372).

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, have also beenprepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222).Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., WO 99/14226).Furthermore, synthesis of 2′-amino-LNA, a novel conformationallyrestricted high-affinity oligonucleotide analog has been described inthe art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Inaddition, 2′-Amino- and 2′-methylamino-LNA's have been prepared and thethermal stability of their duplexes with complementary RNA and DNAstrands has been previously reported.

Certain modified sugar moieties are well known and can be used to alter,typically increase, the affinity of the antisense compound for itstarget and/or increase nuclease resistance. A representative list ofpreferred modified sugars includes but is not limited to bicyclicmodified sugars (BNA's), including LNA and ENA (4′-(CH₂)₂—O-2′ bridge);substituted sugars, especially 2′-substituted sugars having a 2′-F,2′-OCH₃ or a 2′-O(CH₂)₂—OCH₃ substituent group; and 4′-thio modifiedsugars. Sugars can also be replaced with sugar mimetic groups amongothers. Methods for the preparations of modified sugars are well knownto those skilled in the art. Some representative patents andpublications that teach the preparation of such modified sugars include,but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080;5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134;5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; and6,600,032; and WO 2005/121371.

BNAs and 2′ modifications.

In certain embodiments, 2′ modifications that may be assessed include,but are not limited to: halo, allyl, amino, azido, amino, SH, CN, OCN,CF₃, OCF₃, O—, S—, or N(R_(m))-alkyl; O—, S—, or N(R_(m))-alkenyl; O—,S— or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl,O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)) orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m), and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl. These 2′-substituent groups can be furthersubstituted with substituent groups selected from hydroxyl, amino,alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy(S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl where each R_(m)is, independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, 2′ modifications that may be assessed include,but are not limited to F, —NH₂, N₃, O—CH₃, O(CH₂)₃NH₂), CH₂—CH═CH₂,—O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, 2′-O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)),—O(CH₂)₂—O—(CH₂)₂N(CH₃)₂, and N-substituted acetamide(O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m), and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl. Another list of 2′-substituent groupsincludes F, OCF₃, O—CH₃, OCH₂CH₂OCH₃, 2′O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), —O(CH₂)₂—O(CH₂)₂N(CH₃)₂, and N-substitutedacetamides (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, assays of the present invention may be used topredict or assess RNAi activity or potency of a novel compoundcomprising novel modifications.

Certain RNAi Sequences

In certain embodiments, the present invention provides a method ofidentifying sequences with RNA; activity and/or potency. In certain suchembodiments, the sequence comprises a plurality of pyrimidines in theseed region. The “seed region” is the second through the eighthnucleoside counting from the 5′ end of a guide strand of an siRNA or ofa single stranded oligonucleotide RNAi agent. In certain embodiments, asequence having RNAi activity has at least 3, at least 4, at least 5, atleast 6, or at least 7 pyrimidines in the seed region.

The sequence of the PTEN and Eg5 guide strands are5′-UUGUCUCUGGUCCUUACUU-3′ and 5′-AUAGACUUCAUCCUUGUUG-3′ respectively.The PTEN siRNA was a better competitor and had greater Ago2 binding,Ago2 activity, RNAi activity, and RNAi potency than the Eg5 siRNA, eventhough the two siRNAs had the same length and modification motif. Thedifference may be attributable to the greater pyrimidine content of theseed region of the PTEN sequence.

For all oligonucleotide compounds discussed herein, sequence,nucleoside, nucleoside modification, and internucleoside linkage mayeach be selected independently. In certain embodiments, RNAi agents aredescribed by a motif. In such embodiments, any motif may be used withany sequence, whether or not the sequence and/or the motif isspecifically disclosed herein. The sequence listing accompanying thisfiling provides certain nucleic acid sequences independent of chemicalmodification. Though that listing identifies each sequence as either“RNA” or “DNA” as required, in reality, those sequences may be modifiedwith any combination of chemical modifications and/or motifs.

Certain Cells

In certain embodiments, the present invention provides cell basedassays. In such embodiments, suitable cell include, but are not limitedto HeLa, U87, and T47D cells. In certain embodiments, the presentinvention provides a method of selecting a target cell or tissue forRNAi, including, but not limited to RNAi based therapy, comprisingselecting a cell or tissue type with a relatively high concentration ofAgo2.

In certain embodiments, the invention provides cell based assays inwhich an RNAi agent is transfected into a cell, Ago2 binding and/or Ago2activity occurs in the cell and is assessed. In certain embodiments, anRNAi agent is transfected into a cell and binding occurs in the cell andthen cell is lysed allowing the Ago2 bound to the RNAi agent to becollected and assessed in a cell-free system. Thus, in certainembodiments, activity is assessed outside a cell after binding hasoccurred inside a cell. In certain of such embodiments, Ago2 isover-expressed in the cell prior to transfection of the RNAi agent.

In certain embodiments, the invention provides cell free assays. Incertain such embodiments, Ago2 is contacted with an RNAi agent outside acell. In certain such embodiments, the Ago2 is obtained byimmunoprecipitating it from a cell. In certain such embodiments, theAgo2 is overexpressed in the cell prior to immunoprecipitation. Incertain embodiments, the Ago2 is recombinant Ago2. Ago2 that is obtainedby immunoprecipitation is expected to remain in association with certainother factors or proteins such as TRBP. The present inventors have shownthat in certain instances recombinant Ago2 has comparable binding andactivity.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein havebeen described with specificity in accordance with certain embodiments,the following examples serve only to illustrate the compounds describedherein and are not intended to limit the same. Each of the references,GenBank accession numbers, and the like recited in the presentapplication is incorporated herein by reference in its entirety.

EXAMPLES Example 1 Cells and Reagents

Tissue culture medium, trypsin and Lipofectamine2000 were purchased fromInvitrogen (Carlsbad, Calif.). HeLa, T47D and U87-MG cells were obtainedfrom the American Type Tissue Collection (Manassas, Va.) and cultured inDMEM supplemented with 10% fetal calf serum, streptomycin (0.1 μg/ml),and penicillin (100 units/ml). Treatment of cells with siRNA and RNAseHASOs was performed using Opti-MEM (Invitrogen) containing 2-5 μg/mlLipofectamine 2000 and the indicated amount of siRNA/ASO for 3-5 h at37° C., as described previously (Dean and Griffey, Antisense NucleicAcid Drug Dev., 7, 229-233). For the generation of IC₅₀ curves cellswere treated at doses ranging from 2 μM to 60 nM in half-log serialdilutions (N=4-8/dose). IC₅₀ curves and values were generated usingPrism 4 software (GraphPad).

Example 2 Preparation of Antisense Oligonucleotides and siRNAs

Synthesis and purification of phosphorothioate/2′-MOE oligonucleotideswas performed using an Applied Biosystems 380B automated DNAsynthesizer. All ASOs were full phosphorothioate with 2′-β-methoxyethylsubstitutions at positions 1-5 and 16-20 (boldface type). Residues 5-15are unmodified 2′-oligodeoxynucleotides, so they can serve as substratesfor RNase H. The sequences are as follows. Ago2(ISIS 136764),CTGCTGGAATGTTTCCACTT; Dicer (ISIS 138648), GCTGACCTTTTTGCTTCTCA; TRBP(ISIS 237288), TGCGGTGGGCTGGCCCAGAC; Exportin5 (ISIS 350560),GTTACCATTCTGTACAGGTA. Silencer Pre-designed siRNAs for knockdown of RISCproteins were obtained from Ambion (Austin, Tex.). Ago2: siRNA ID#133832; Dicer: siRNA ID# 137011; Exportin-5: siRNA ID# 109277; TRBP:siRNA ID# 139948. PTEN and Eg5 synthetic unmodified siRNAs werepurchased from Dharmacon Research, Inc (Boulder, Colo.). siRNA duplexeswere formed according to the manufacture's instructions. In brief, 1.6μl of a 250 μM antisense stock was combined with 1.6 μl of a 250 μMsense stock, 4 μl of 5× universal buffer (500 mM potassium acetate, 150mM HEPES-KOH, pH 7.4, 10 mM magnesium acetate) and 12.8 μl of ultrapurewater followed by heating at 90 C for one minute. The reaction was thenallowed to cool to ambient temperature. The final concentration of theduplex was 20 μM in 1× universal buffer (100 mM potassium acetate, 30 mMHEPES-KOH, pH 7.4, 2 mM magnesium acetate). The sequence of the PTENsiRNA is AAGTAAGGACCAGAGACAA (sense) and TTGTCTCTGGTCCTTACTT(antisense). The sequence of the Eg5 siRNA is CAACAAGGATGAAGTCTAT(sense)and ATAGACTTCATCCTTGTTG (antisense).

Example 3 Taqman RT-PCR

Quantitative RT-PCR was performed essentially as described elsewhere.See Winer, et al., Anal. Biochem., 270, 41-19 (1999). Briefly, 200 ng oftotal RNA was analyzed in a final volume of 50 containing 200 nMgene-specific PCR primers, 0.2 mM of each dNTP, 75 nM fluorescentlylabeled oligonucleotide probe, 5 μl RT-PCR buffer, 5 mM MgCl2, 2 U ofPlatinum TaqDNA Polymerase (Invitrogen Life Technologies), and 8 U ofRNase inhibitor. Reverse transcription was performed for 30 min at 48°C. followed by PCR: 40 thermal cycles of 30 s at 94° C. and 1 minute at60° C. using an ABI Prism 7700 Sequence Detector (Applied Biosystems).The sequence for the human Ago2 primer/probe set used in the RT-PCRreaction was CCAGCTACACTCAGACCAACAGA for the forward primer,GAAAACGGAGAATCTAATAAAATCAATGAC for the reverse primer andCGTGACAGCCAGCATCGAACATGAGA for the probe. The sequence for the humanDicer primer/probe set used in the RT-PCR reaction wasATTAACCTTTTGGTGTTTGATGAGTGT for the forward primer,GCGAGGACATGATGGACAAATT for the reverse primer andATCTTGCAATCCTAGACCACCCCTATCGAGAA for the probe. The sequence for thehuman TRBP primer/probe set used in the RT-PCR reaction wasCAGCCCACCGCAAAGAAT for the forward primer, TGCCACTCCCAATCTCAATG for thereverse primer and CACCATGACCTGTCGAGTGGAGCGT for the probe. The sequencefor the human Exportin 5 primer/probe set used in the RT-PCR reactionwas GCTGTGAATATTCTCGGTTTGATTT for the forward primer,GGAAGCTAGTTTTGGGATCCAA for the reverse primer andTCCTCCCGAGCACAACAAGGAGAGG for the probe.

Example 4 Western Blotting

Whole cell extracts were prepared by lysing cells inradioimmunoprecipitation assay (RIPA) buffer (1×PBS, 1% Nonidet P-40,0.1% deoxycholate, and 0.1% SDS containing complete protease inhibitormix (Boehringer Mannheim). Protein concentration of the cell extractswas measured by Bradford assay (no. 500-0201; Bio-Rad). Equal amounts ofprotein (10-20 μg) were resolved on a NuPAGE Novex 8-16% Tris-glycinegel in Tris-Gly SDS running buffer (Invitrogen Life Technologies) andtransferred to polyvinylidene difluoride membranes (Invitrogen LifeTechnologies). The membranes were blocked for 1 hour in TBS containing0.05% Tween 20 (TBST) and 5% milk powder. After overnight incubation at4° C. with a 1/1000 dilution of a mouse monoclonal antibody to Ago2,Exportin 5 (Abnova), Dicer [13D6] (Abeam), or a 1/3000 dilution of arabbit polyclonal antibody to TRBP, the membranes were washed in PBScontaining 0.05% Tween 20 and incubated with a 1/5000 dilution of goatanti-rabbit or goat anti-mouse HRP-conjugated Ab in blocking buffer.Membranes were washed and developed using ECL detection system (AmershamBiosciences). Subsequently, membranes were blocked for 2 h at roomtemperature in TBST plus 5% milk powder. After incubation at roomtemperature with a 1/5000 dilution of a mouse monoclonal tubulin Ab (no.T-5 168; Sigma-Aldrich), the membranes were washed in PBS containing0.1% Tween 20 and incubated with a 1/5000 dilution of goat anti-mouseHRP-conjugated Ab in blocking buffer and developed as detailed above andexposed to film (Kodak).

Example 5 RISC Protein Overexpression and Reduction

Plasmids expressing Dicer and Exportin 5 under the control of the CMVpromoter were obtained from OriGene Technologies, Inc. (Rockville, Md.).cDNAs for Ago2 and TRBP were isolated from HeLa cell cDNA by PCR andcloned into pcDNA3.1 (Invitrogen). For RISC gene overexpression assays,10 ug of plasmid was introduced into cells at 50% confluence in 10-cmdishes using SuperFect Reagent (Qiagen). Following a 3 hour treatment,plasmid was removed and fresh DMEM added to the cells. Following anovernight incubation cells were trypsinized then seeded in 96 wellplates at 4000-6000 cells per well. Cells were allowed to adhere for 4hours, then siRNAs were added in the presence of Lipofectamine 2000reagent as detailed above. For siRNA competition experiments both siRNAswere premixed then added simultaneously to the cells. All siRNA/ASOtreatments were performed in triplicate or quadruplicate. Following the5 hour transfection, siRNAs were aspirated and fresh DMEM added to thecells. Treated cells were incubated overnight. The next day total RNAwas purified from 96-well plates using an RNeasy 3000 BioRobot (Qiagen,Valencia, Calif.). Reduction of target mRNA expression was determined byreal time RT-PCR using an ABI Prism 7700 Sequence Detector (AppliedBiosystems, Foster City, Calif.). The sequence for the human Eg5primer/probe set used in the RT-PCR reaction is GCCCCAAATGTGAAAGCATT forthe forward primer, CTAAAGTGGGCTTTTTGTGAACTCT for the reverse primer andCCTTTAAGAGGCCTAACTC for the probe. The sequence for the human PTENprimer/probe set is AATGGCTAAGTGAAGATGACAAT for the forward primer,TGCACATATCATTACACCAG-TTCGT for the reverse primer andAGATGCCGTGTTTGATGGCTCCAGC for the probe. mRNA levels were normalized tototal RNA for each sample as measured by Ribogreen (Invitrogen).

For RISC reduction, HeLa cells were seeded in 10 cm dishes at 650,000cells per plate. The following day RISC specific siRNA/ASO (above) wasadded at 50 nM in OptiMEM media in the presence of 5 μg/ml Lipofectamine2000. Following a 5 hour incubation the transfection mixture wasaspirated and DMEM added to the cells. The remainder of the experimentwas carried out as described for the RISC overexpression studies above.

Example 6 Kinetic Analysis of siRNA Competition

To analyze kinetics of RISC loading HeLa cells were seeded in 96 wellplates at 4500/well. The following day cells were transfected with 300μM Eg5 siRNA using Opti-MEM media containing 5 μg/ml Lipofectamine 2000.The Eg5/Lipofectamine 2000 complex was removed at timepoints between 0and 240 minutes from the initiation of the transfection and replacedwith a mixture of 300 pM Eg5 siRNA and 10 nM PTEN siRNA (N=4/timepoint).Cells were incubated 4 hours, then siRNA removed and fresh DMEM added.Cells were incubated overnight then total RNA was purified and Eg5 mRNAexpression evaluated by qRT/PCR.

For RISC unloading kinetics HeLa cells were seeded in 96 well plates ata density of 4000 cells/well. The following day cells were transfectedwith 10 nM PTEN competitor siRNA in Opti-MEM media containing 3 μg/mlLipofectamine 2000. After 3 hours the PTEN/Lipofectamine 2000 complexwas removed, the cells washed with PBS, and fresh DMEM+5% FCS added. 20nM complexed with 5 μg/ml Lipofectamine 2000, then 1/20 volume addeddirectly to the pretreated cells (final concentration=1 nM) Immediatelyfollowing the PTEN siRNA pretreatment and at intervals from 2-18 hoursEg5 siRNA was transfected at 300 pM as detailed above (N=8/timepoint).For each timepoint the siRNA/lipid complex was removed after 3 hours,cells washed with PBS, and fed with DMEM+10% FCS. Cells were incubatedovernight then total RNA was purified and Eg5 mRNA expression evaluatedby qRT/PCR. Where indicated the experiment was performed in the presenceof 25 μg/ml cycloheximide (Sigma). Inhibition of protein synthesis wasconfirmed by measurement of cellular incorporation ³⁵S-Translabel (MPBiomedicals).

For kinetic analysis of siRNA activity HeLa cells were seeded in 96 wellplates then treated with 10 nM Eg5 siRNA as detailed above. Cells wereharvested and total RNA isolated beginning at 15 minutes from theinitiation of transfection. The transfection mixture was removed fromcells at 4 hours for the 7 and 18 hour timepoints and complete mediaadded. Eg5 and PTEN mRNA expression was assessed by qRT/PCR andnormalized to total RNA as measured by ribogreen assay.

Example 7 Levels of siRNA Components in Various Cell Lines

Previous work has suggested that RISC components may be rate limiting asco-transfection of two siRNAs resulted in loss of activity (Koller etal., Nucleic Acid Research, 34:16, 4467-4476 (2006)). Ago2 has beenshown to be required for siRNA activity in mammalian cells. Sontheimer,Nat Rev Mol Cell Biol., 6(2):127-38 (2005). Liu, et al., Science, 305,1437-1441 (2004). Although Dicer and TRBP are clearly important andinvolved in the siRNA pathway, whether they are required for theactivities of siRNAs is somewhat controversial. Two reports in whichsiRNAs were used to reduce Dicer and/or TRBP suggested that Dicer andTRBP were required for siRNA activity (Doi, et al., Curr. Biol., 13,41-46 (2003) and Chendrimada, Nature 436, 740-44 (2005)), but studies inwhich the Dicer gene was knocked out suggested that Dicer was requiredfor miRNA processing and activity, but not required for siRNA activity.See Kanellopoulou, et al., Genes Dev., 19 489-501 (2005); Murchison, etal., Proc. Natl. Acad. Sci. USA, 102, 12135-12140 (2005). It has beendemonstrated that TRBP is required for the recruitment of Ago2 to thesiRNA bound by Dicer (Chendrimada, Nature 436, 740-44 (2005)). Inaddition, it has recently been shown that the Exportin-5-based exclusionof siRNAs from the nucleus can, when Exportin-5 itself is inhibited,become a rate-limiting step for siRNA induced silencing activity. SeeOhrt, et al., Nucleic Acids Res., 34, 1369-1380 (2006). In an attempt tocorrelate RISC levels with observed differences in siRNA competition andactivity the expression levels of these genes were characterized in HeLacells, an epithelial carcinoma cell line, T47D, a ductal carcinoma cellline, and U87-MG, and a glioblastoma cell line.

The relative expression levels of the messenger RNAs for the RISCrelated genes were measured by quantitative RT/PCR. The data wasanalyzed by comparing the levels of each mRNA to the levels in HeLacells in order to estimate differences in relative levels of each mRNAbetween cell lines. While HeLa and U87-MG cells were found to havesimilar levels of Dicer and TRBP mRNA, levels of the same messages were5-7 fold higher in T47Ds. Ago2 and Exportin-5 mRNAs were found to bereduced in U87-MG cells relative to HeLa and T47D cells which hadroughly equivalent amounts of the same mRNAs.

The levels of Ago2, Dicer, TRBP and exportin-5 proteins were assessed byWestern blot. Dicer and TRBP levels were comparable in U87-MG and HeLacells, and Ago2 and Exportin-5 were lower in U87-MG cells, in agreementwith the messenger RNA data. RISC expression was also evaluated in othercells lines and found to vary considerably. Of the cell lines tested,the highest levels of Ago2 were found in HeLa cells. T47D cellsexpressed Dicer and TRBP at the highest levels, while the lowest levelsof Ago2 and Exportin-5 were found in U87-MGs.

Example 8 The Magnitude of PTEN and Eg5 siRNA Competition in VariousCell Lines

It has previously been shown that an siRNA targeted to PTEN is aneffective competitor of an siRNA targeted to Eg5 (Koller et al., NucleicAcid Research, 34:16, 4467-4476 (2006)). To determine if siRNAcompetition could be correlated with differences in expression of thevarious RISC proteins, the activity of Eg5 siRNA was evaluated in theabsence or presence of increasing amounts of PTEN siRNA in HeLa, T47D,and U87-MG cells. Cells were transfected with Eg5 siRNA at doses rangingbetween 20 pM and 20 nM alone or co-transfected with the PTEN competitorsiRNA at doses of 2, 6, or 20 nM for 4 hours as detailed herein.Following transfection, cells were incubated overnight then harvestedand total RNA isolated. Quantitative RT/PCR was performed to assess thereduction of Eg5 mRNA. In the absence of competitor siRNA (▪), the IC₅₀for siRNA mediated reduction of Eg5 was similar in HeLa and T47D cells(92±18 and 76±15 pM respectively) and approximately 2 fold higher inU87-MGs (198±45 pM). In all cell lines the IC₅₀'s for Eg5 inhibitionincreased when co-transfected with increasing amounts of PTEN competitorsiRNA. In HeLa cells, competition with PTEN siRNA at the highestconcentration of 20 nM (A) resulted in an increase in the IC₅₀ for Eg5siRNA to 772±227 pM, approximately 8 fold higher than observed in theabsence of competitor (FIG. 1A). Similarly, in T47Ds, competition with20 nM PTEN siRNA increased the IC₅₀ nearly 7 fold (FIG. 14B). Incontrast, a similar magnitude of competition was achieved in U87 cellsusing only 6 nM PTEN siRNA competitor (; IC₅₀=1.2±0.3 nM) and an over20 fold increase in IC₅₀ was observed in the presence of 20 nMcompetitor (IC₅₀=4.4±2.1 nM, FIG. 1C). These data illustrate that themagnitude of PTEN and Eg5 competition varies between cell lines.

Example 9 Comparison Ago2, Dicer, TRBP, and Exportin-5 Levels and siRNAActivity and Magnitude of Competition

To determine if reduction in native levels of RISC components mightresult in changes in siRNA potency and competition, reduction of RISCcomponents was effected using siRNA or RNaseH-dependent antisenseoligonucleotide (ASO) inhibitors in HeLa cells. The reduction of eachcomponent and the evaluation of the activities of siRNAs in a singlecell line eliminates any contribution of cell line to cell linevariation in transfection efficiency which might result in apparentdifferences in RNA potency and competition. As shown in FIG. 2A, HeLacells treated with either siRNA (S) or antisense (H) inhibitors showed amarked decrease in the targeted RISC mRNAs 24 hours after the initiationof transfection.

The effects of Ago2 or Dicer protein reduction on siRNA competition andpotency were initially evaluated in HeLa cells. Cells were treated withtarget specific siRNAs as detailed herein. 24 hours later cells wereseeded in 96-well plates and IC₅₀s for Eg5 siRNA were assessed in thepresence or absence of 15 nM PTEN siRNA competitor. Reductions in Ago2and Dicer protein levels were confirmed at the time of Eg5 siRNAtransfection by Western blot analysis (FIG. 3A). In cells withoutreduction of RISC components (FIG. 3B, top panel) the IC₅₀ for Eg5 siRNAin the absence of PTEN competitor was 40±8 pM (solid line), while in thepresence of competitor the IC₅₀ was determined to be 285±50 pM (dottedline). Reduction of Dicer (middle panel) had little effect on the siRNAactivity or competition in HeLa cells with IC₅₀s in the absence andpresence of PTEN competitor of 50±8 pM and 300±40 pM respectively. Incontrast, in cells in which Ago2 was reduced (lower panel), there was asignificant change in both siRNA potency and competition. The IC₅₀ inthe absence of PTEN competitor was determined to be 241±45 pM, whilecompetition with PTEN siRNA in the same cells resulted in an IC₅₀ of2.98±0.7 nM. Comparing the Eg5 IC₅₀s in the absence of PTEN competition,there was a decrease in potency of approximately 6 fold in the cellstreated with Ago2 ASO. In addition, the magnitude of PTEN competitionwas increased in these cells as the IC₅₀ in the presence of competitorwas approximately 12 fold greater than in the absence of competitor,compared to approximately 7 fold in HeLa cells with normal Ago2 levels.Therefore, the magnitude of competition was almost 2 fold greater inHeLa cells in which Ago2 levels are reduced. It is also interesting tonote that Ago2 reduction resulted in a change in siRNA potency andcompetition comparable to that observed in U87 cells and after reductionof Ago2 with siRNAs or ASOs the level of Ago2 in HeLa cells appears tobe roughly comparable to the level in untreated U87 cells (compare with(FIG. 3C Table I).

In another experiment; HeLa cells were treated with siRNAs targeted toexportin-5 or Ago2. Reduction of targeted protein was confirmed after 24hours by Western blot analysis (FIG. 3C). Reduction of exportin-5 had noeffect on siRNA potency as the IC₅₀s for Eg5 reduction were nearly thesame in both untreated (FIG. 3D, top panel) and exportin-5 reduced(middle panel) HeLas. The increases in IC₅₀'s in the presence of PTENsiRNA competitor were also nearly the same for untreated and exportin-5reduced HeLa cells (compare dotted lines). Once again, in cells in whichAgo2 was reduced (lower panel) there was a significant decrease inpotency of Eg5 siRNA alone as the IC₅₀ increased from 61±7 pM inuntreated HeLa cells to 357±49 pM in Ago2-reduced HeLa cells (comparesolid curves). The magnitude of competition by the PTEN siRNA competitorwas also increased in Ago2 reduced cells. In untreated HeLa cells, anapproximately 3 fold increase in Eg5 siRNA IC₅₀ was observed in thepresence of PTEN siRNA competitor. In contrast, the Eg5 siRNA IC₅₀ inthe presence of PTEN siRNA was determined to be 2.9±0.9 nM, anapproximate 8 fold increase in IC₅₀. In other experiments TRBP wastargeted, and reduction of this protein had no effect on siRNAcompetition or potency while Ago2 reduction repeatedly and consistentlyresulted in decreases in siRNA potency and competition using either ASOsor siRNAs to effect protein reduction.

The effect of RISC protein overexpression on siRNA competition was alsoevaluated. As shown FIG. 2B, cells transfected with expression plasmidsshowed significant increases in Dicer, TRBP, Ago2, or exportin-5 protein24 hours after the initiation of transfection. U87-MG control and Ago2overexpressing cells were transfected with Eg5 siRNA at doses from 2 pMto 60 nM in the presence or absence of 5 nM PTEN siRNA competitor. Thefollowing day total RNA was isolated and IC₅₀s determined by qRT/PCR.Ago2 mRNA levels as determined by qRT/PCR also confirmed overexpressionof the message in cells harboring the plasmid. Increased Ago2 expressionin U87-MG cells resulted in an increase in siRNA potency with the IC₅₀for Eg5 mRNA reduction decreasing from 291±79 pM in untreated U87-MGcells to 65±18 pM in cells overexpressing Ago2 (Table I). The IC₅₀ inthe presence of PTEN siRNA increased in the untreated cells byapproximately 5.6 fold to 1.6±0.5 nM. In the Ago2 transfected U87-MGcells the IC₅₀ was 138±40 pM, a 2.1 fold increase over the IC₅₀ obtainedin cells with normal protein levels. Overexpression of the otherproteins (FIG. 2B) had no effect on the potency of Eg5 siRNA orcompetition due to PTEN siRNA.

These data illustrated that Ago2 levels, but not Dicer, TRBP, orExportin-5 levels, limit siRNA activity and the magnitude of siRNAcompetition.

Example 10 PTEN siRNA is a More Effective Competitor than Eg5 siRNA

It has previously been observed that there is, in general, a correlationbetween siRNA potency and competition, with more potent siRNAs beingmore effective competitors. (Koller et al., Nucleic Acid Research,34:16, 4467-4476 (2006)). Having demonstrated that siRNA competition isdependant upon the availability of Ago2 within the cell, it washypothesized that when Ago2 is limiting the siRNA with the greateraffinity for Ago2 would be the more effective competitor. To test thishypothesis, competitive inhibition of Ago2 binding was employed tomeasure the relative binding affinities of the Eg5 and PTEN siRNAs asdetailed herein. The K_(D) for the PTEN sense strand was determined tobe 92±17 nM (FIG. 4A, squares) while the K_(D) for Eg5 was determined tobe 671±138 nM. This approximately seven-fold difference in bindingaffinity was observed in separate experiments.

Competition between the same siRNAs was compared in U87 cells since theyhave the lowest Ago2 levels of the cell lines characterized. Cells weretransfected PTEN siRNA ranging between 20 μM and 20 nM alone or in thepresence of Eg5 competitor siRNA at doses of 0, 2, 6, or 20 nM asdetailed above. IC₅₀ curves for PTEN siRNA alone and in the presence ofEg5 competitor are shown in FIG. 4B. The IC₅₀ for PTEN siRNA in theabsence of competition was determined to be significantly lower (31±10pM) than that of Eg5 siRNA alone (198±45 pM). In contrast to the strongcompetition observed when PTEN siRNA was used as a competitor of Eg5siRNA with the IC₅₀ for Eg5 increasing by over 20 fold in cells treatedwith 20 nM PTEN siRNA (FIG. 1C, open triangles), when Eg5 siRNA was usedas a competitor of PTEN siRNA only 5 fold change in IC₅₀ at the 20 nMdose of competitor (FIG. 4B). Eg5 siRNA was also a less effectivecompetitor than PTEN at the 2 and 6 nM doses (inverted triangle andcircles respectively).

These data illustrate that PTEN siRNA is a more effective competitorthan Eg5 siRNA.

Example 11 Kinetics of RISC Loading and Unloading

siRNA competition was used to evaluate the kinetics of RISC loading andunloading. To evaluate RISC loading, Eg5 siRNA activity was measured inthe presence of the PTEN competitor siRNA added simultaneously or atvarious times after the initiation of transfection of the Eg5 siRNA. Inthe absence of PTEN siRNA competition, a 78.3±11.2% reduction of Eg5message was observed in cells treated with 300 pM Eg5 siRNA, while inthe presence of 10 nM PTEN siRNA competitor added simultaneously only40.6±11.5% reduction was observed. When the addition of PTEN competitorsiRNA was delayed from 5 to 30 minutes no effect on competition wasobserved. When addition of PTEN siRNA was delayed by 60 minutes thepercent reduction of Eg5 siRNA increased to 65.8±12.5% and by 120minutes no competition was observed with the percent reduction returningto levels comparable to those observed with Eg5 siRNA treatment alone.These results indicate that the Eg5 siRNA loaded and saturated the freeRISC available in the cells in 1-2 hours. Once RISC was loaded thesiRNA/RISC, the more potent PTEN siRNA, the guide strand of whichdisplays higher affinity for purified Ago2, was unable to compete.

To evaluate unloading of RISC, HeLa cells were pre-treated for 3 hourswith 15 nM PTEN siRNA. Cells were washed, then 300 pM Eg5 siRNA added attimes following PTEN siRNA treatment. Eg5 siRNA transfections werecarried out for 3 hours, then the cells incubated overnight. Thefollowing day RNA was purified and the percent reduction of Eg5 mRNAdetermined by qRT/PCR. Cells that were not pre-treated with PTEN siRNA(E) showed greater than 65% reduction of Eg5 mRNA (FIG. 5B). Incontrast, cells pre-treated with PTEN siRNA displayed approximately 28%reduction of Eg5 mRNA at the same dose when the Eg5 siRNA wastransfected immediately after PTEN siRNA treatment (T=0). After 2 hoursthe percent reduction was increased from 28% approximately 46% andincreased to 55% by 9 hours. After 12 hours, no PTEN siRNA competitionwas observed with levels of Eg5 reduction returning to over 65%. Todetermine if the loss of competition resulted from unloading of RISC orbecause of new synthesis of Ago2, the effects of inhibiting proteinsynthesis in HeLa cells were determined in similar experiments.Cycloheximide (25 μg/mL) was added to cells following the PTEN siRNApre-treatment. When cells were treated with cycloheximide, Eg5 mRNAreduction followed the same pattern as in the absence of cycloheximide(FIG. 5B, crosshatched bars). In the absence of competition almost 70%reduction of Eg5 mRNA was observed. At T=0 15 nM PTEN siRNA resulted in42% reduction of Eg5 and by T=12 hours had returned to Eg5 siRNA-onlylevels. In these experiments, S³⁵ methionine incorporation wasdetermined to be inhibited by greater than 95%. The similarity ofrecovery of Eg5 inhibition in the presence or absence of cycloheximidesuggests that new protein synthesis is not required. Instead, RISC mustin some manner be unloaded and recycled.

Together, these kinetic data indicate that RISC is loaded fairly rapidlywithin 2 hours of the initiation of transfection. Target cleavage,product release and RISC recycling is a slower process taking as long as12 hours to complete. The kinetics of mRNA reduction by Eg5 siRNA wereexplored to determine if mRNA reduction followed a similar time courseas RISC loading and recycling. HeLa cells were transfected with 10 nMEg5 siRNA as detailed herein. Cells were harvested and Eg5 mRNAreduction determined beginning 15 minutes after the initiation oftransfection. A slight reduction in Eg5 mRNA levels (FIG. 6, stripedbars) was observed after 1 hour. By 2 hours Eg5 levels had been reducedby 35% and by 4 hours 76%. Maximal messenger RNA reduction was notobserved until between 7 and 18 hours after the initiation of siRNAtreatment. No Eg5 mRNA reduction was observed in untreated cells (FIG.6, solid bars).

Example 12 Ago2 Activity Assay in Cells

Synthetic oligonucleotides were manufactured by Dharmacon Research, Inc.siRNA duplexes were formed according to the manufacturer's instructions.The final concentration of the duplex was 20 mM in 100 mM potassiumacetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate. The RNAsubstrates were 5′-end-labeled with 32P using 10 U of T4 polynucleotidekinase. The labeled oligoribonucleotide was purified on a 12% denaturingpolyacrylamide gel. The specific activity of the labeled oligonucleotidewas approximately 3000 to 8000 cpm/fmol.

The cDNA of human Ago2 with an N-terminal HA epitope was subcloned intothe mammalian expression vector phCMV-2. HeLa cells (CCL-2) were treatedwith the Ago2 expression plasmid using Effectene transfection reagent.The cells were then treated with 75 nM synthetic oligoribonucleotides orsiRNAs using Lipofectamine 2000 reagent 24 h after the treatment withAgo2 expression plasmid and incubated for 18 h. Cells were harvestedwith Trypsin, then washed twice with 1 mL of cold PBS. The cell pelletwas re-suspended in 500 mL of lysis buffer (150 mM NaCl, 0.5% NP-40, 2mM MgCl2, 2 mM CaCl2, 20 mM Tris at pH 7.5, protease inhibitor and 1 mMDTT), and passed through an Insulin Syringe. Supernatants were clarifiedwith a 10,000 g clearing spin for 10 min. and protein concentrationswere determined. 1.8 mg protein was incubated with 15 ul of HA II beads(Covance, Calif.) for 2 h, washed 3× with lysis buffer and equilibratedin cleavage buffer (10 mM Tris at pH 7.5, 100 mM KCl, 2 mM MgCl2,Protease Inhibitor. 0.5 mM DTT). 0.1 nM 32P labeled target RNA was addedand cleavage reactions quenched at specified times in gel loading buffer(Ambion, Tex.). Cleavage reactions were analyzed by denaturing PAGE andquantitated with Storm 850 Phosphorimager (Molecular Dynamics).

Example 13 Cell-Free Assay Using Immunoprecipitated Ago2 (Post-LoadAssays)

To assess Ago2 activity in a cell-free assay, immunoprecipitates wereprepared as above (Example 12) using cells treated with the Ago2expression plasmid but not treated with synthetic oligoribonucleotidesor siRNAs. Instead, immunoprecipitate equilibrated in cleavage bufferwas incubated for 2 h with either the synthetic oligoribonucleotides orsiRNAs. Immunoprecipitate was washed with cleavage buffer and 0.1 nM 32Plabeled target RNA was added. Cleavage reactions were quenched andanalyzed as above.

To assess Ago2 binding in a cell-free assay, immunoprecipitates wereprepared as above for the cell-free activity assay except that 0.18 mgtotal protein was used. 0.1 nM 32P labeled antisense strand wasincubated with the Ago2 immunoprecipitate in the presence of increasingconcentrations of cold antisense strand or competitor nucleic acidconstruct for 2 h. The immunoprecipitate was washed in cleavage bufferto remove unbound nucleic acids. Binding reactions were stopped by theaddition of scintillation cocktail and bound radioactivity analyzed in ascintillation counter. Bound radioactivity was plotted as a function ofthe cold competitor concentration and dissociation constants weredetermined from the non-linear regression fit of the data.

Example 14 Assays Using Recombinant Ago2

The full length of hAgo2 was first sub-cloned into the pGEX-3× vector.The GST-Ago2 fusion DNA was further sub-cloned into the baculovirusshuttle vector pENTR2B. Ago2 was expressed in Sf9 insect cells usingBaculoDirect Baculovirus Expression System. Sf9 cells infected with hightiter virus were harvested and the cells were gently lysed in PBScontaining 0.5% NP-40, 1 mM DTT and proteases inhibitor and thencentrifuged. The pellet was re-suspended and subject to sonication andre-centrifugation. Supernatants were GST affinity purified followed byHPLC purification. The purified GST-Ago2 protein yielded puritiesgreater than 95%.

Ago2 activity assays and Ago2 binding assays using recombinant Ago2 wereperformed as above (Example 13) for immunoprecipitated Ago2. Binding andactivity for various siRNAs were found to be similar usingimmunoprecipitated Ago2 and recombinant Ago2, despite the fact thatrecombinant Ago2 is not in association with other cellular factors, suchas TRBP, which one might expect to influence binding and/or activity inthe immunoprecipitated Ago2.

1. A method of determining relative potency of a chemically-synthesizedRNAi agent comprising: forming a first test sample comprising thechemically-synthesized RNAi agent and Ago2; forming a second test samplecomprising an RNAi control agent and Ago2; and measuring the degree ofbinding between the chemically-synthesized RNAi agent and the Ago2protein, and the degree of binding between the control RNAi agent andAgo2, wherein the difference in the degree of binding of thechemically-synthesized RNAi agent and degree of binding of the controlRNAi agent to Ago2 indicates potency of the chemically-synthesized RNAiagent relative to the control RNAi agent.
 2. The method of claim 1,wherein the Ago2 is recombinant.
 3. The method of claim 1, wherein theAgo2 is obtained by immunoprecipitation from a cell.
 4. The method ofclaim 3, wherein the cell is made to over-express the Ago2 protein. 5.The method of claim 1, wherein the degree of binding is measured bymeasuring an amount of chemically-synthesized RNAi agent bound to theAgo2.
 6. The method of claim 1, wherein the chemically-synthesized RNAiagent is an oligonucleotide.
 7. The method of claim 6, wherein theoligonucleotide is from about 15 to about 27 nucleotides in length. 8.The method of claim 6, wherein the oligonucleotide comprises acomplementary strand from about 15 to about 27 nucleotides in length. 9.The method of claim 6, wherein the oligonucleotide is an antisenseoligoribonucleotide.
 10. The method of claim 6, wherein theoligonucleotide comprises at least one sugar-modified nucleotide. 11.The method of claim 10, wherein the sugar-modified nucleotide is a 2′substituted nucleotide.
 12. The method of claim 6, wherein theoligonucleotide comprises at least one modified internucleoside linkage.13. The method of claim 12, wherein the modified internucleoside linkageis a phosphorothioate internucleoside linkage.
 14. A method ofdetermining relative potency of a chemically-synthesized RNAi agentcomprising: forming a first test sample comprising thechemically-synthesized RNAi agent and Ago2; forming a second test samplecomprising an RNAi control agent and Ago2; and measuring the Ago2activity of the chemically-synthesized RNAi agent and the Ago2 activityof the RNAi control agent, wherein the difference in the Ago2 activityof the chemically-synthesized RNAi agent and Ago2 activity of thecontrol RNAi agent indicates potency of the chemically-synthesized RNAiagent relative to the control RNAi agent.
 15. The method of claim 14,wherein the Ago2 is recombinant.
 16. The method of claim 14, wherein theAgo2 is obtained by immunoprecipitation from a cell.
 17. The method ofclaim 15, wherein the cell is made to over-express the Ago2 protein. 18.The method of claim 14, wherein the degree of binding is measured bymeasuring an amount of chemically-synthesized RNAi agent bound to theAgo2.
 19. The method of claim 14, wherein the chemically-synthesizedRNAi agent is an oligonucleotide.
 20. The method of claim 19, whereinthe oligonucleotide is from about 15 to about 27 nucleotides in length.21. The method of claim 19, wherein the oligonucleotide comprises acomplementary strand from about 15 to about 27 nucleotides in length.22. The method of claim 19, wherein the oligonucleotide is an antisenseoligoribonucleotide.
 23. The method of claim 19, wherein theoligonucleotide comprises at least one sugar-modified nucleotide. 24.The method of claim 23, wherein the sugar-modified nucleotide is a 2′substituted nucleotide.
 25. The method of claim 19, wherein theoligonucleotide comprises at least one modified internucleoside linkage.26. The method of claim 25, wherein the modified internucleoside linkageis a phosphorothioate internucleoside linkage.
 27. A method ofdetermining relative potency of a chemically-synthesized RNAi agentcomprising: forming a first test sample comprising a control RNAi agentand Ago2 and a second test sample comprising the control RNAi agent, thechemically-synthesized RNAi agent, and Ago2; and measuring the Ago2activity or binding of the control RNAi agent in the first and secondtest samples, wherein the amount of decrease in the Ago2 activity orbinding of the control RNAi agent in the presence of the chemicallysynthesized RNAi agent indicates potency of the chemically-synthesizedRNAi agent relative to the control RNAi agent.
 28. The method of claim27, wherein the Ago2 is recombinant.
 29. The method of claim 27, whereinthe Ago2 is obtained by immunoprecipitation from a cell.
 30. The methodof claim 29, wherein the cell is made to over-express the Ago2 protein.31. The method of claim 27, wherein the degree of binding is measured bymeasuring an amount of chemically-synthesized RNAi agent bound to theAgo2.
 32. The method of claim 27, wherein the chemically-synthesizedRNAi agent is an oligonucleotide.
 33. The method of claim 32, whereinthe oligonucleotide is from about 15 to about 27 nucleotides in length.34. The method of claim 32, wherein the oligonucleotide comprises acomplementary strand from about 15 to about 27 nucleotides in length.35. The method of claim 32, wherein the oligonucleotide is an antisenseoligoribonucleotide.
 36. The method of claim 32, wherein theoligonucleotide comprises at least one sugar-modified nucleotide. 37.The method of claim 36, wherein the sugar-modified nucleotide is a 2′substituted nucleotide.
 38. The method of claim 32, wherein theoligonucleotide comprises at least one modified internucleoside linkage.39. The method of claim 38, wherein the modified internucleoside linkageis a phosphorothioate internucleoside linkage.
 40. A method ofdetermining the relative potency of two or more chemically-synthesizedRNAi agents comprising: forming a first test sample comprising a controlRNAi agent, a first chemically-synthesized RNAi agent, and Ago2; forminga second test sample comprising the same control RNAi agent, a secondchemically-synthesized RNAi agent, and Ago2; and measuring the Ago2activity or binding of the control RNAi agent in the first and secondtest samples, wherein the difference in the Ago2 activity or binding ofthe control RNAi agent in the first and second test samples indicatespotency of the first chemically-synthesized RNAi agent and the secondchemically-synthesized RNAi agent, relative to one another.